A group of low-molecular-weight GTP-binding proteins (G-proteins) with molecular weights of 20,000-30,000 with no subunit structures is observed in organisms. To date, over fifty or more members have been found as the super family of the low-molecular-weight G-proteins in a variety of organisms, from yeast to mammals. The low-molecular-weight G-proteins are divided into four families of Ras, Rho, Rab and the others based on homologies of amino acid sequences. It has been revealed that the small G-proteins control a variety of cellular functions. For example, the Ras protein is considered to control cell proliferation and differentiation, and the Rho protein is considered to control cell morphological change, adhesion and motility.
The Rho protein, having GDP/GTP-binding activity and intrinsic GTPase activity, is believed to be involved in cytoskeletal responses to extracellular signals such as lysophosphatidic acid (LPA) and certain growth factors. When the inactive GDP bound form of Rho is stimulated, it is transformed to the active GTP bound form of Rho protein (hereinafter referred to as "the activated Rho protein") by GDP/GTP exchange proteins such as Smg GDS, Dbl or Ost. The activated Rho protein then acts on target proteins to form stress fibers and focal contacts, thus inducing the cell adhesion and motility (Experimental Medicine, Vol. 12, No. 8, 97-102 (1994); Takai, Y. et al., Trends Biochem. Sci., 20, 227-231 (1995)). On the other hand, the intrinsic GTPase activity of the Rho protein transforms the activated Rho protein to the GDP bound form of Rho protein. This intrinsic GTPase activity is enhanced by what is called GTPase-activating proteins (GAP) (Lamarche, N. & Hall, A. et al., TIG, 10, 436-440 (1994)).
Natural RhoA proteins bear Cys-A-A-Leu (SEQ ID NO: 6) (A: aliphatic amino acid) on the C-terminal thereof. The Cys residue is geranylgeranylated by geranylgeranyl transferase and its carboxyl group methylated in posttranslational processing, which is considered essential for the binding of the Rho protein to cell membranes and interaction with activity regulating proteins as well as for the expression of the functions thereof (Imazumi, K. et al., Experimental Medicine, Vol. 13, 646-656 (1995)).
The Rho family proteins, including RhoA, RhoB, RhoC, Rac1, Rac2 and Cdc42, share more than 50% sequence identity with each other. The Rho family proteins are believed to be involved in inducing the formation of stress fibers and focal contacts in response to extracellular signals such as lysophosphatidic acid (LPA) and growth factors (A. J. Ridley & A. Hall, Cell, 70, 389-399 (1992); A. J. Ridley & A. Hall, EMBO J., 1353, 2600-2610 (1994)). The subfamily Rho is also considered to be implicated in physiological functions associated with cytoskeletal rearrangements, such as cell morphological change (H. F. Parterson et al., J. Cell Biol., 111, 1001-1007 (1990)), cell adhesion (Morii, N. et al., J. Biol. Chem., 267, 20921-20926 (1992); T. Tominaga et al., J. Cell Biol., 120, 1529-1537 (1993); Nusrat, A. et al., Proc. Natl. Acad. Sci. USA, 92, 10629-10633 (1995)*; Landanna, C. et al., Science, 271, 981-983 (1996)*, cell motility (K. Takaishi et al., Oncogene, 9, 273-279 (1994), and cytokinesis (K. Kishi et al., J. Cell Biol., 120, 1187-1195 (1993); I. Mabuchi et al., Zygote, 1, 325-331 (1993)). (An asterisk hereinafter indicates a publication issued after the first filed application which provides the right of the priority of the present application.) In addition, it has been suggested that the Rho is involved in the regulation of smooth muscle contraction (K. Hirata et al., J. Biol. Chem., 267, 8719-8722 (1992); M. Noda et al., FEBS Lett., 367, 246-250 (1995); M. Gong et al., Proc. Natl. Acad. Sci. USA, 93, 1340-1345 (1996)*), and the expression of phosphatidylinositol 3-kinase (PI3 kinase) (J. Zhang et al., J. Biol. Chem., 268, 22251-22254 (1993)), phosphatidylinositol 4-phosphate 5-kinase (PI 4,5-kinase) (L. D. Chong et al., Cell, 79, 507-513 (1994)) and c-fos (C. S. Hill et al., Cell, 81, 1159-1170 (1995)).
Recently, it has also be found that Ras-dependent tumorigenesis is suppressed when the Rho protein of which the amino acid sequence has been partly substituted is introduced to cells, revealing that the Rho protein plays an important role in Ras-induced transformation, that is, tumorigenesis (G. C. Prendergast et al., Oncogene, 10, 2289-2296 (1995); Khosravi-Far, R. et al., Mol. Cell. Biol., 15, 6443-6453 (1995)*; R. Qiu et al., Proc. Natl. Acad. Sci. USA, 92, 11781-11785 (1995)*; Lebowitz, P. et al., Mol. Cell, Biol., 15, 6613-6622 (1995)*).
It has also been proved that the Rho protein enhances not only cell proliferation, cell motility and cell aggregation but also smooth muscle contraction. Recent studies have demonstrated that the Rho protein is involved in the smooth muscle contraction (K. Hirata et al., J. Biol. Chem., 267, 8719-8722 (1992); Noda, M. et al., FEBS Lett., 367, 246-250 (1995)). Therefore, it can reasonably be assumed that the activated Rho-binding protein is also involved in the smooth muscle contraction.
The phosphorylation of myosin light chain plays vital roles in the smooth muscle contraction (Kamm, K. E. & Stull, J. T., Annu. Rev. Pharmacol. Toxicol., 25, 593-603 (1985); Hartshorne, D. J. & Johnson, D. R. (1987) in Physiology of the Gastrointestinal Tract, (Johnson, L. R., ed), pp. 423-482, Raven Press, New York; Sellers, J. R. & Adelstein, R. S. in The Enzyme (Boyer, P. and Erevs, E. G., eds), Vol. 18, pp. 381-418, Academic Press, San Diego, Calif. (1987)) and the actin-myosin interaction for stress fiber formation in non-muscle cells (Huttenlocher, A. et al., Curr. Opi. Cell Biol., 7, 697-706 (1995)) and, thus, involved in cytokinesis and cell motility (Huttenlocher, A. et al., Curr. Opi. Cell Biol., 7, 697-706 (1995)).
Myosin light chain kinase phosphorylates primarily the Ser-19 of myosin light chain (Kamm, K. E. & Stull, J. T., Annu. Rev. Pharmacol. Toxicol., 25, 593-603 (1985); Hartshorne, D. J. & Johnson, D. R., (1987) in Physiology of the Gastrointestinal Tract, (Johnson, L. R., ed), pp. 423-482, Raven Press, New York; Sellers, J. R. & Adelstein, R. S. in The Enzyme (Boyer, P. and Erevs, E. G., eds), Vol. 18, pp. 381-418, Academic Press, San Diego, Calif. (1987); Ikebe, M. & Hartshorne, D. J., J. Biol. Chem., 260, 10027-10031 (1985)). No protein kinase obtained thus far other than specific kinases such as myosin light chain kinase phosphorylates this site (Tan, J. L. et al., Annu. Rev. Biochem., 61, 721-759 (1992).
When a smooth muscle is stimulated by an agonist such as an angiotonic, Ca.sup.2+ moves into cytoplasm, and activates the calmodulin-dependent myosin light chain kinase. The phosphorylated myosin light chain induces myosin-actin interaction, which in turn activates myosin ATPase (Kamm, K. E. & Stull, J. T., Annu. Rev. Pharmacol. Toxicol., 25, 593-603 (1985); Hartshorne, D. J. & Johnson, D. R., (1987) in Physiology of the Gastrointestinal Tract, (Johnson, L. R., ed), pp. 423-482, Raven Press, New York; Sellers, J. R. & Adelstein, R. S. in The Enzyme (Boyer, P. and Erevs, E. G., eds), Vol. 18, pp. 381-418, Academic Press, San Diego, Calif. (1987)), thus inducing the smooth muscle contraction (Kamm, K. E. & Stull, J. T., Annu. Rev. Pharmacol. Toxicol., 25, 593-603 (1985); Hartshorne, D. J. & Johnson, D. R., (1987) in Physiology of the Gastrointestinal Tract, (Johnson, L. R., ed), pp. 423-482, Raven Press, New York; Sellers, J. R. & Adelstein, R. S. in The Enzyme (Boyer, P. and Erevs, E. G., eds), Vol. 18, pp. 381-418, Academic Press, San Diego, Calif. (1987)). However, Ca.sup.2+ level in the cytosol is not necessarily proportional to the contraction level, indicating another explanation for the mechanism of the regulation of Ca.sup.2+ sensitivity in smooth muscle contraction (Bradley, A. B. & Morgan, K. G., J. Physiol., 385, 437-448 (1987)). As GTP.gamma.S (non-hydrolyzable GTP analog) decreases the Ca.sup.2+ concentration necessary for the contraction of permeabilized (skinned) smooth muscles, GTP-binding proteins were expected to regulate Ca.sup.2+ sensitivity (Kitazawa, T. et al., Proc. Natl. Acad. Sci. U.S.A., 88, 9307-9310 (1991); Moreland, S. et al., Am. J. Physiol., 263, 540-544 (1992)). The Rho protein was proved to be involved in Ca.sup.2+ sensitivity of smooth muscles, which is enhanced by GTP (Hirata, K. et al., J. Biol. Chem., 267, 8719-8722 (1992)). Recently, in permeabilized smooth muscles, it was demonstrated that GTP.gamma.S enhances the phosphorylation of myosin light chain at submaximal Ca.sup.2+ concentration, suggesting that the enhancement was attributed to the activation of the Rho protein and the inhibition of the enzymatic activity of myosin light chain phosphatase, which dephosphorylates myosin light chain (Noda, M. et al., FEBS Lett., 367, 246-250 (1995)). However, it has not been resolved yet how the Rho protein inhibits myosin light chain phosphatase, whether the enhancement of the phosphorylation of myosin light chain by the Rho protein is attributed solely to the inhibition of myosin light chain phosphatase activity, and, thus, how the Rho protein regulates the Ca.sup.2+ sensitivity of smooth muscles and enhances the smooth muscle contraction.
These findings indicate that the Rho protein controls a variety of signal transduction pathways for cell morphological change, cell adhesion, cell motility, cytokinesis, tumorigenesis, metastasis, vascular smooth muscle contraction, etc. The Rho protein appears to be able to act on a number of target molecules to control all these signal transduction pathways.
It is only recently (after the first filed application which provides the right of the priority of the present application) that a several proteins have been reported as candidates of Rho-targets in mammals: protein kinase N (PKN) (Watanabe, G. et al., Science, 271, 645-648 (1996)*; Amano, M. et al., Science, 271, 648-650 (1996)*), rhophilin (Watanabe, G. et al., Science, 271, 645-648 (1996)*, citron (Madaule, P. et al., FEBS Lett., 377, 243-248 (1995)*), ROK.alpha. (Leung, T. et al., J. Biol. Chem., 270, 29051-29054 (1995)*), p160.sup.ROCK (Ishizaki, T. et al., EMBO J., 15, 1885-1893 (1996)*) and rhotekin (Reid, T. et al., J. Biol. Chem., 271, 13556-13560 (1996)*). All these proteins bind to GTP-binding RhoA protein, except that citron binds also to GTP-binding Rac1.
Among these proteins, PKN has an enzymatic region which closely resembles the protein kinase region of protein kinase C and exhibits serine/threonine kinase activity (Mukai, H. & Ono, Y., Biochem. Biopys. Res. Commun., 199, 897-904 (1994); Mukai, H. et al., Biochem. Biopys. Res. Commun., 204, 348-356 (1994)). On the other hand, ROK.alpha. (Leung, T. et al. (1995), ibid.) and p160.sup.ROCK (Ishizaki, T. et al. (1996)*, ibid.) also have amino acid sequences resembling a serine/threonine kinase region (Leung, T. et al. (1995)*, ibid.).
In addition to those reported in mammals, protein kinase C1 (PKC1) in yeast (Saccharomyces cerevisiae) has recently been identified as a target protein of the Rho1 protein, corresponding to RhoA in mammals (Nonaka, H. et al., EMBO J., 14, 5931-5938 (1995)*). Only recently, 1,3-.beta.-glucan synthase has been identified as a target protein of the Rho1p protein in yeast (Saccharomyces cerevisiae) (Drgonova, J. et al., Science, 272, 277-279 (1996)*; Qadota, H. et al., Science, 272, 279-281 (1996)*).
However, mechanisms of intercellular signal transduction involving the activated Rho protein, particularly those of tumorigenesis and smooth muscle contraction, are still unknown.
An asterisk hereinafter indicates a publication issued after the first filed application which provides the right of the priority of the present application.